This research was supported in part by the NHLBI Intramural Research Program (DIR HL-006247). We would like to thank the NIH Mouse Imaging Facility for guidance in image acquisition and analysis.

All methods described here have been approved by the Institutional Animal Care and Use Committee (ACUC) of the National Heart Lung and Blood Institute.
1. Preparation
<ol>
	<li>Inject the mouse intraperitoneally with heparin (1 unit/g mouse body weight) and allow it to ambulate for 2 min.</li>
	<li>Euthanize the animal in a CO2 chamber.</li>
	<li>Arrange the mouse in a supine position on a surgical board and secure all four limbs to the board with tape. Use magnification for fine dissection.</li>
</ol>
2. Exposing lungs and trachea
<ol>
	<li>Spray the ventral side of the mouse with 70% ethanol to minimize hair interference.</li>
	<li>Grasp the abdominal skin with forceps and make a small incision with scissors in the umbilical region. Slide the tips of scissors into the fascial layer between the abdominal musculature and skin and begin separating the two layers. Work rostrally, removing the skin from the abdomen, ribcage, and neck.</li>
	<li>Open the abdominal musculature with scissors and cut laterally on both sides until the diaphragm is exposed.</li>
	<li>Gently grasp the xiphoid process and slightly lift the ribcage maximizing the view of the caudal lungs through the thin, semitransparent diaphragm. Carefully make a small incision in the diaphragm just beneath the xiphoid process. The lungs will collapse and retract away from the diaphragm. Dissect the diaphragm away from the ribcage, taking care not to nick the lung parenchyma.</li>
	<li>Locate and sever the inferior vena cava (IVC) and esophagus where they pass through the diaphragm. Use gauze to clean up any pooling blood in the thoracic cavity, avoiding contact with the lungs.</li>
	<li>Grasp the xiphoid once again and gently lift. Cut the ribcage bilaterally (roughly at the midaxillary line) avoiding contact with the lungs. Remove the anterior ribcage entirely, making the final cut along the sternal angle just before the manubrium.</li>
	<li>Using a prefilled syringe, liberally wet the lungs with phosphate-buffered saline (PBS, pH 7.4) to prevent drying out. Continue this routine throughout the procedure.</li>
	<li>Using forceps, grasp the manubrium and gently elevate away from the body. Using scissors, cut 1-2 mm lateral to the manubrium, severing clavicles, and remove. This will expose the thymus underneath.</li>
	<li>Grasp each lobe of the thymus, pull apart, and remove. Repeat this procedure with the submandibular gland. Finally, remove the muscular tissue overlaying the trachea. 
	NOTE: Following the dissection, the heart, ascending aorta (AA), pulmonary arterial trunk (PAT), and trachea should be visible. Ensure the primary arterial branches off the trunk are not divided or injured.</li>
</ol>
3. PA catheterization and blood perfusion
<ol>
	<li>To assemble Unit 1, thread 15 cm of PE-10 tubing onto the hub of a 30 G needle and attach to a 1 mL syringe prefilled with 10-4 M sodium nitroprusside (SNP) in PBS. Prime the tubing by advancing the plunger until all the air is purged from this unit ( Figure 1). 
	CAUTION: SNP is toxic if swallowed. Avoid contact with the skin and eyes. Wash skin thoroughly after handling. Wear appropriate personal protective equipment.
	<ol>
		<li>Alternatively, assemble Unit 2. For mice postnatal day 7 (P7) and younger, use a hemostat to detach an additional 30 G needle from its hub and thread the needle onto the open end of the tubing of Unit 1 (Figure 1).</li>
	</ol>
	</li>
	<li>Instead of a needle, use curved sharp forceps to grasp one end of a 10 cm length of 7-0 silk. Penetrate the apex of the heart entering from one side and passing the tips of the forceps through the muscle and out of the other side. Grasp the silk with another set of forceps and pull approximately a 2 cm length through and tie off. Take the remaining 8 cm end of the suture, tugging the heart caudally, and tape the end to the surgical board. 
	NOTE: This will create tension, further exposing the great vessels and tethering the heart in place, allowing for easier placement of the catheter in the pulmonary artery.</li>
	<li>Hook the tips of curved forceps under both the AA and PAT. Pull a 3 cm length of 7-0 silk back through the opening and create a single-throw loose suture.</li>
	<li>Using scissors make a 1-2 mm incision toward the apex of the heart, penetrating the thin-walled right ventricle (RV), to allow for the insertion of the catheter (Unit 1). Prior to the insertion, confirm there is no air in the system. Introduce the primed tubing into the right ventricle and gently advance into the semitransparent thin-walled PAT.
	<ol>
		<li>Visually verify that the catheter has not advanced into either the left or right pulmonary branches and does not abut the pulmonary artery branchpoint. Using tape, secure the distal portion of the tubing to the surgical board. 
		NOTE: To identify the RV, use forceps to pinch the right side of the heart. Unlike the left ventricle, the relatively thin free wall of the RV should be easily grasped.</li>
		<li>For mice younger than P7, attach Unit 2 to a micromanipulator and introduce the needle end of the unit into the PAT as described above using the manipulator.</li>
	</ol>
	</li>
	<li>Gently tighten the loose suture around both great vessels and cut the 8 cm length of suture created in step 3.2 to return the heart to a natural resting position. The catheter is now firmly secured within the PAT.</li>
	<li>Clip the left auricle of the heart to allow perfusate to exit the system.</li>
	<li>Secure SNP-containing syringe (Unit 1 or Unit 2, size dependent) in the syringe pump and perfuse the solution at a rate of 0.05 mL/min to flush the blood and maximally dilate the vasculature. Blood/perfusate will exit via the clipped auricle. Continue perfusion until perfusate runs clear (~200 &#181;L in an adult mouse, less for younger animals). 
	NOTE: When perfusing the low viscosity PBS/SNP, a relatively higher infusion rate was used in the interest of saving time. The more viscous polymer compound is infused at a slower rate to prevent overfilling, rupture, and maximize control over distal endpoints.</li>
</ol>
4. Tracheostomy and lung inflation
<ol>
	<li>Construct the lung inflation unit (Figure 2).
	<ol>
		<li>Connect a flexible plastic 24 G intravenous (IV) catheter (needle removed)/butterfly infusion set to a stopcock, attached to an open 50 mL syringe (no plunger). Hang the syringe from a ring stand.</li>
		<li>Add 10% buffered formalin to the syringe. Open the stopcock, allowing formalin to enter the tubing and purge all air from the system. Close the stopcock and raise the syringe until the meniscus is 20 cm above the trachea8. 
		CAUTION: Formalin is flammable, carcinogenic, acutely toxic when ingested, and causes skin irritation, serious eye damage, skin sensitization, and germ cell mutagenicity. Avoid ingestion and contact with skin and eyes. Avoid inhalation of the vapor or mist. Keep away from sources of ignition. Wear appropriate personal protective equipment.</li>
	</ol>
	</li>
	<li>Place two loose sutures inferior to the cricoid cartilage 2-4 mm apart.</li>
	<li>Using scissors, make a small incision in the cricothyroid ligament superior to the sutures.</li>
	<li>Insert the IV catheter into the opening and advance the tip beyond the two loose sutures.</li>
	<li>Tighten the sutures around the trachea and open the stopcock. Allow the formalin to enter the lungs by gravity and wait for 5 min for the lungs to fully inflate. If the lungs adhere to the ribcage during inflation, grasp the outside of the ribcage with blunt tipped forceps and move in all directions to assist in freeing the lobes. Do not make direct contact with lungs.</li>
	<li>After 5 min, back the IV catheter beyond the first suture and ligate. Repeat for the second suture. The lungs are now inflated in a closed, pressurized state.</li>
</ol>
5. Casting the vasculature
<ol>
	<li>&#160;In a 1.5 mL tube, prepare 1 mL of an 8:1:1 solution8 of polymer:diluent:curing agent and gently invert several times to ensure good mixing.</li>
	<li>Remove the plunger from a 1 cc syringe, cover the opposite end with a gloved finger, and pour the polymer compound into the syringe. Carefully reinsert the plunger, invert, and advance the plunger to remove all air and form a meniscus at the tip of the syringe.</li>
	<li>Remove the SNP/PBS syringe from the hub of the needle and drip additional PBS into the hub to create a meniscus. Carefully check the hub for trapped air, dislodge if necessary, and reform the meniscus. Join the hub to the syringe filled with the polymer compound. 
	NOTE: Creating a meniscus on both ends significantly reduces the chance for air to enter the system.</li>
	<li>Attach the polymer compound filled syringe to the syringe pump and infuse at 0.02 mL/min. 
	NOTE: For smaller lungs, a slower rate can be helpful to prevent overfilling but, is not essential.</li>
	<li>Monitor the compound as it freely moves down the PE tubing and note the syringe volume as it enters the PAT. Continue filling until all lobes are filled completely down to the capillary level and stop the syringe pump. Check the syringe volume again. 
	NOTE: After several runs an estimated volume can be used to gauge an approximate endpoint (~35 &#956;L for an adult mouse and ~5 &#181;L for a P1 pup). After the pump is halted, the residual pressure in the system will continue to push the polymer compound into the pulmonary arteries. All lung lobes should fill at a similar rate.</li>
	<li>Cover the lungs with a fiber optic cleaning wipe, liberally apply PBS, and allow the carcass to sit undisturbed for 30-40 min at room temperature. During this period, the polymer compound will cure and harden.</li>
	<li>Remove the catheter, sever the arms/lower half of the mouse, and place the head/thorax into a 50 mL conical filled with 10% buffered formalin overnight.</li>
	<li>After fixation, grasp the trachea and gently separate the heart/lung unit from the remaining rib cage and thorax. Place the heart/lung block in a formalin filled scintillation vial. Discard the rest.</li>
</ol>
6. Alternative vascular beds for casting (Table 1)
NOTE: Each target vascular bed may require different catheter placements, infusion rates, and optimal filling times. Thus, multiple animals will be necessary to cast multiple organs.
<ol>
	<li>For systemic vascular beds superior or inferior to the diaphragm follow steps 1.1-2.5 as above. See additional notes on the portal system and diaphragm (Table 1).</li>
	<li>Grasp the xiphoid process with a hemostat and cut the ribcage bilaterally (roughly in the midaxillary line) just prior to the internal thoracic arteries.</li>
	<li>Fold the still connected ribcage over such that it is resting on the animal&#39;s neck/head, fully exposing the chest cavity.</li>
	<li>Follow step 3.1 above, then remove the lungs. Once the thoracic aorta (TA) is visible, hook the tips of curved forceps underneath it, ~10 mm superior to the diaphragm. Grasp a 3 cm length of 7-0 silk, pull back through the opening under the TA, and create a single-throw loose suture. Repeat this procedure ~8 mm above the diaphragm.</li>
	<li>For structures superior to the diaphragm, use spring scissors to create a small hole (~30% of the total circumference) on the ventral portion of the TA, ~2 mm inferior to the loose sutures placed in step 6.4.
	<ol>
		<li>For structures inferior to the diaphragm, instead, create a small hole ~2 mm superior to the loose sutures.</li>
	</ol>
	</li>
	<li>Depending on the animal size, introduce Unit 1 or 2 into the vessel, advance beyond loose sutures, and gently ligate the vessel.</li>
	<li>Follow step 3.7, setting the syringe pump at a rate of 1.0 mL/min and perfusing a minimum of 5 mL. Perfusate will exit via the IVC.</li>
	<li>Follow steps 5.1 - 5.4 adjusting the infusion rate to 0.05 mL/min, visually monitoring the target tissue in real-time. 
	NOTE: Infusion volume will be organ and animal age specific. The volume can be further limited by ligating arterial branches leading to non-target vascular beds (i.e., brain, liver, kidney, intestine).</li>
	<li>Follow 5.6 then remove target tissue and place in formalin.</li>
</ol>
7. Sample mount, scan, and reconstruction for micro-CT
<ol>
	<li>Using paraffin film, create a flat surface on the scanning bed and center the wet sample on this surface (Figure 3A). 
	NOTE: If motion artifact is detected, the sample may require further stabilization.</li>
	<li>Lightly tent/cover sample with additional paraffin film to prevent dehydration. Take special care not to rest the paraffin film on the sample causing deformation to the tissue (Figure 3B).</li>
	<li>Scan the sample using settings outlined in Table 2 and standardize these parameters within a given experiment. 
	NOTE: This is experiment/endpoint dependent. Standardize the chosen parameters for the ease of comparison between samples.</li>
	<li>Transfer the reconstructed scans for post-processing and analysis.</li>
</ol>

The authors have nothing to disclose

Executed properly, this method yields striking images of pulmonary arterial networks, allowing for comparison and experimentation in rodent models. Several critical steps along the way ensure success. First, investigators must heparinize the animal in the preparatory stage to prevent blood clots from forming in the pulmonary vasculature and chambers of the heart. This allows for the complete arterial transit of polymer compound. Second, when puncturing the diaphragm and removing the ribcage, take care to protect the lung...

The focus of this technique is the visualization of pulmonary arterial architecture using a polymer-based compound in mice. While extensive work has been performed on systemic vascular beds such as brain, heart, and kidney1,2,3,4,5, less information is available regarding the preparation and filling of the pulmonary arterial network. The aim of this study, therefore, is to expand upon previous work6,7,8 and provide a detailed written and visual reference that investigators can easily follow to produce high-resolution images of the pulmonary arterial tree.
While numerous methods exist for labeling and imaging lung vasculature, such as magnetic resonance imaging, echocardiography, or CT angiography9,10, many of these modalities fail to adequately fill and/or capture the small vessels, limiting the scope of what can be studied. Methods such as serial sectioning and reconstruction provide high resolution but are time/labor-intensive11,12,13. Surrounding soft tissue integrity is compromised in traditional corrosion casting10,13,14,15,16. Even animal age and size become factors when attempting to introduce a catheter or, the resolution is lacking. The polymer injection technique, on the other hand, fills arteries to the capillary level and when combined with &#181;CT, allows for unparalleled resolution5. Samples from mouse lungs as young as postnatal day 14 have been successfully casted8 and processed in a matter of hours. These can be rescanned indefinitely, or even sent for histological preparation/electron microscopy (EM) without compromising the existing soft tissue17. The main limitations to this method are the upfront cost of CT equipment/software, challenges with accurately monitoring intravascular pressure, and the inability to acquire data longitudinally in the same animal.
This paper builds on existing work to further optimize the pulmonary artery injection technique and push age/size related boundaries down to postnatal day 1 (P1) to yield striking results. It is most useful for teams that want to study arterial vascular networks. Accordingly, we provide new guidance for catheter placement/stabilization, increased control over fill rate/volume, and highlight notable pitfalls for increased casting success. Resulting casts can then be used for future characterization and morphologic analysis. Perhaps more importantly, this is the first visual demonstration, to our knowledge, that walks the user through this intricate procedure.

<table border="0" cellpadding="0" cellspacing="0" style="width:829px;" width="828">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">1cc syringe</td>
			<td style="width:135px;">Becton Dickinson</td>
			<td style="width:117px;">309659</td>
			<td style="width:346px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">20ml Glass Scintillation Vials</td>
			<td style="width:135px;">Fisher</td>
			<td>03-340-25P</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">30G Needle</td>
			<td style="width:135px;">Becton Dickinson</td>
			<td style="width:117px;">305106</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">50mL conical tubes</td>
			<td style="width:135px;">Cornin</td>
			<td style="width:117px;">352098</td>
			<td>For sample Storage and scanning</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">60cc syringe</td>
			<td style="width:135px;">Becton Dickinson</td>
			<td style="width:117px;">309653</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">7-0 silk suture</td>
			<td style="width:135px;">Teleflex</td>
			<td style="width:117px;">103-S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Analyze 12.0 Software</td>
			<td style="width:135px;">AnalyzeDirect Inc.</td>
			<td style="width:117px;">N/A</td>
			<td>Primary Software</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Amira 6.7 Software</td>
			<td style="width:135px;">Thermo Scientific</td>
			<td style="width:117px;">N/A</td>
			<td>Alternative Sofware</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">CeramaCut Scissors 9cm</td>
			<td style="width:135px;">Fine Science tools</td>
			<td style="width:117px;">14958-09</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Ceramic Coated Curved Forceps</td>
			<td style="width:135px;">Fine Science tools</td>
			<td style="width:117px;">11272-50</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:231px;">CO2 Tank</td>
			<td style="width:135px;">Robert&#39;s Oxygen Co.</td>
			<td style="width:117px;">n/a</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Dual syringe pump</td>
			<td style="width:135px;">Cole Parmer</td>
			<td style="width:117px;">EW-74900-10</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Dumont Mini-Forceps</td>
			<td style="width:135px;">Fine Science tools</td>
			<td style="width:117px;">11200-14</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Ethanol</td>
			<td style="width:135px;">Pharmco</td>
			<td style="width:117px;">111000200</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:231px;">Formalin</td>
			<td style="width:135px;">Sigma - Life Sciences</td>
			<td style="width:117px;">HT501128</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Gauze</td>
			<td style="width:135px;">Covidien</td>
			<td style="width:117px;">441215</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:22px;width:231px;">Hemostat</td>
			<td style="width:135px;">Fine Science tools</td>
			<td style="width:117px;">13013-14</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:231px;">Heparin (1000USP Units/ml)</td>
			<td style="width:135px;">Hospira</td>
			<td style="width:117px;">NDC 0409-2720-01</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:231px;">Horos Software</td>
			<td style="width:135px;">Horos Project</td>
			<td style="width:117px;">N/A</td>
			<td>Alternative Sofware</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">induction chamber</td>
			<td style="width:135px;">n/a</td>
			<td style="width:117px;">n/a</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Kimwipe</td>
			<td style="width:135px;">Fisher</td>
			<td style="width:117px;">06-666</td>
			<td>fiber optic cleaning wipe</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Labelling Tape</td>
			<td style="width:135px;">Fisher</td>
			<td style="width:117px;">15966</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Magnetic Base</td>
			<td style="width:135px;">Kanetec</td>
			<td style="width:117px;">N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Micro-CT system</td>
			<td style="width:135px;">SkyScan&#160;</td>
			<td style="width:117px;">1172</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Microfil (Polymer Compound)</td>
			<td style="width:135px;">Flowech Inc.</td>
			<td style="width:117px;">Kit B - MV-122</td>
			<td>8 oz. of MV compound; 8 oz. of diluent; MV-Curing Agent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Micromanipulator</td>
			<td style="width:135px;">Stoelting</td>
			<td style="width:117px;">56131</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Monoject 1/2 ml Insulin Syringe</td>
			<td style="width:135px;">Covidien</td>
			<td style="width:117px;">1188528012</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Octagon Forceps Straight Teeth</td>
			<td style="width:135px;">Fine Science tools</td>
			<td style="width:117px;">11042-08</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:231px;">Parafilm</td>
			<td style="width:135px;">Bemis company, Inc.</td>
			<td style="width:117px;">#PM999</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">PE-10 tubing</td>
			<td style="width:135px;">Instech</td>
			<td style="width:117px;">BTPE-10</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Phospahte buffered Saline</td>
			<td style="width:135px;">BioRad</td>
			<td style="width:117px;">#161-0780</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Ring Stand</td>
			<td style="width:135px;">Fisher</td>
			<td style="width:117px;">S13747</td>
			<td>Height 24in.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Sodium Nitroprusside</td>
			<td style="width:135px;">sigma</td>
			<td style="width:117px;">71778-25G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Steel Plate</td>
			<td style="width:135px;">N/A</td>
			<td style="width:117px;">N/A</td>
			<td>16 x 16 in. area, 1/16 in thick</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:231px;">Straight Spring Scissors</td>
			<td style="width:135px;">Fine Science tools</td>
			<td style="width:117px;">15000-08</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:231px;">SURFLO 24G Teflon I.V. Catheter</td>
			<td style="width:135px;">Santa Cruz Biotechnology</td>
			<td style="width:117px;">360103</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Surgical Board</td>
			<td style="width:135px;">Fisher</td>
			<td>12-587-20</td>
			<td>This is a converted slide holder</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Universal 3-prong clamp</td>
			<td style="width:135px;">Fisher</td>
			<td style="width:117px;">S24280</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Winged Inf. Set 25X3/4, 12&#34; Tubing</td>
			<td style="width:135px;">Nipro</td>
			<td style="width:117px;">PR25G19</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Zeiss Stemi-508 Dissection Scope</td>
			<td style="width:135px;">Zeiss</td>
			<td style="width:117px;">n/a</td>
			<td></td>
		</tr>
	</tbody>
</table>

vascular casting of adult and early postnatal mouse lungs for micro-ct imaging

Blood vessels form intricate networks in 3-dimensional space. Consequently, it is difficult to visually appreciate how vascular networks interact and behave by observing the surface of a tissue. This method provides a means to visualize the complex 3-dimensional vascular architecture of the lung.
To accomplish this, a catheter is inserted into the pulmonary artery and the vasculature is simultaneously flushed of blood and chemically dilated to limit resistance. Lungs are then inflated through the trachea at a standard pressure and the polymer compound is infused into the vascular bed at a standard flow rate. Once the entire arterial network is filled and allowed to cure, the lung vasculature may be visualized directly or imaged on a micro-CT (&#181;CT) scanner.
When performed successfully, one can appreciate the pulmonary arterial network in mice ranging from early postnatal ages to adults. Additionally, while demonstrated in the pulmonary arterial bed, this method can be applied to any vascular bed with optimized catheter placement and endpoints.

A successful cast will exhibit uniform filling of the entire pulmonary arterial network. We demonstrate this in C57Bl/6J mice ranging in age: Postnatal day P90 (Figure 4A), P30 (Figure 4B), P7 (Figure 4C), and P1 (Figure 4D). By controlling the rate of flow and visually monitoring the fill in real-time, reliable endpoints of the most distal vasculature were achieved (<st...

Watch this Scientific Journal Video about Vascular Casting of Adult and Early Postnatal Mouse Lungs for Micro-CT Imaging at JoVE.com

Vascular Casting of Adult and Early Postnatal Mouse Lungs for Micro-CT Imaging

The aim of this technique is ex vivo visualization of pulmonary arterial networks of early postnatal and adult mice through lung inflation and injection of a radio-opaque polymer-based compound via the pulmonary artery. Potential applications for casted tissues are also discussed.

Blood vessels form intricate networks in 3-dimensional space. Consequently, it is difficult to visually appreciate how vascular networks interact and ...

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7.3K Views.  National Institutes of Health. The aim of this technique is ex vivo visualization of pulmonary arterial networks of early postnatal and adult mice through lung inflation and injection of a radio-opaque polymer-based compound via the pulmonary artery. Potential applications for casted tissues are also discussed.

Video: Vascular Casting of Adult and Early Postnatal Mouse Lungs for Micro-CT Imaging

Organotypic Slice Cultures for Studies of Postnatal Neurogenesis

Here we describe a technique for studying hippocampal postnatal neurogenesis in the rodent brain using the organotypic slice culture technique. This method maintains the characteristic topographical morphology of the hippocampus while allowing direct application of pharmacological agents to the developing hippocampal dentate gyrus. Additionally, slice cultures can be maintained for up to 4 weeks and thus, allow one to study the maturation process of newborn granule neurons. Slice cultures allow for efficient pharmacological manipulation of hippocampal slices while excluding complex variables such as uncertainties related to the deep anatomic location of the hippocampus as well as the blood brain barrier. For these reasons, we sought to optimize organotypic slice cultures specifically for postnatal neurogenesis research.

Here we describe a technique for studying hippocampal postnatal neurogenesis using the organotypic slice culture technique. This method allows for in vitro manipulation of adult neurogenesis and allows for the direct application of pharmacological agents to the cultured hippocampus.

Here we describe a technique for studying hippocampal postnatal neurogenesis in the rodent brain using the organotypic slice culture technique. This ...

Methods for Precisely Localized Transfer of Cells or DNA into Early Postimplantation Mouse Embryos

Manipulation and culture of early mouse embryos is a powerful yet largely under-utilized technology enhancing the value of this model system. Conversely, cell culture has been widely used in developmental biology studies. However, it is important to determine whether in vitro cultured cells truly represent in vivo cell types. Grafting cells into embryos, followed by an assessment of their contribution during development is a useful method to determine the potential of in vitro cultured cells. In this study, we describe a method for grafting cells into a defined site of early postimplantation mouse embryos, followed by ex vivo culture. We also introduce an optimized electroporation method that uses glass capillaries of known diameter, allowing precise localization and adjustment of the number of cells receiving exogenous DNA with both high transfection efficiency and low cell death. These techniques, which do not require any specialized equipment, render experimental manipulations of the gastrulation and early organogenesis-stage mouse embryo possible, allowing analysis of commitment in cultured cell subpopulations and the effect of genetic manipulations in situ on cell differentiation.

We demonstrate a method for grafting cultured cells into defined sites of early mouse embryos to determine their in vivo potential. We also introduce an optimized electroporation method that uses glass capillaries of known diameter, allowing the precise delivery of exogenous DNA into a few cells in the embryos.

Manipulation and culture of early mouse embryos is a powerful yet largely under-utilized technology enhancing the value of this model system. Conversely, ...

Assessment of Maternal Vascular Remodeling During Pregnancy in the Mouse Uterus

The placenta mediates the exchange of factors such as gases and nutrients between mother and fetus and has specific demands for supply of blood from the maternal circulation. The maternal uterine vasculature needs to adapt to this temporary demand and the success of this arterial remodeling process has implications for fetal growth. Cells of the maternal immune system, especially natural killer (NK) cells, play a critical role in this process. Here we describe a method to assess the degree of remodeling of maternal spiral arteries during mouse pregnancy. Hematoxylin and eosin-stained tissue sections are scanned and the size of the vessels analysed. As a complementary validation method, we also present a qualitative assessment for the success of the remodeling process by immunohistochemical detection of smooth muscle actin (SMA), which normally disappears from within the arterial vascular media at mid-gestation. Together, these methods enable determination of an important parameter of the pregnancy phenotype. These results can be combined with other endpoints of mouse pregnancy to provide insight into the mechanisms underlying pregnancy-related complications.

This protocol describes a technique to assess changes in the maternal vasculature during pregnancy in mice. Using stereological methods, remodeling of the decidual spiral arteries is assessed quantitatively and the results confirmed qualitatively using immunohistochemistry.

The placenta mediates the exchange of factors such as gases and nutrients between mother and fetus and has specific demands for supply of blood from the ...

Loss- and Gain-of-function Approach to Investigate Early Cell Fate Determinants in Preimplantation Mouse Embryos

Gene silencing and overexpression techniques are instrumental for the identification of genes involved in embryonic development. Direct target gene modification in preimplantation embryos provides a means to study the underlying mechanisms of genes implicated in, for instance, cellular differentiation into the trophectoderm (TE) and the inner cell mass (ICM). Here, we describe a protocol that examines the role of neogenin as an authentic receptor for initial cell fate determination in preimplantation mouse embryos. First, we discuss the experimental manipulations that were used to produce gain and loss of neogenin function by microinjecting neogenin cDNA and shRNA; the effectiveness of this approach was confirmed by a strong correlation between the pair-wise expression levels of either red fluorescent protein (RFP) or green fluorescent protein (GFP) and the immunocytochemical quantification of neogenin expression. Secondly, overexpression of neogenin in preimplantation mouse embryos leads to normal ICM development while neogenin knockdown causes the ICM to develop abnormally, implying that neogenin could be a receptor that relays extracellular cues to drive blastomeres to early cell fates. Given the success of this detailed protocol in investigating the function of a novel embryonic developmental stage-specific receptor, we propose that it has the potential to aid in exploration and identification of other stage-specific genes during embryogenesis.

The goal of this protocol is to describe a loss- and gain-of function method that is applicable to identify neogenin as a stage-specific receptor that leads to trophectoderm and inner cell mass differentiation in preimplantation mouse embryos.

 Gene silencing and overexpression techniques are instrumental for the identification of genes involved in embryonic development. Direct target gene ...

Imaging Cleared Embryonic and Postnatal Hearts at Single-cell Resolution

Single clonal tracing and analysis at the whole-heart level can determine cardiac progenitor cell behavior and differentiation during cardiac development, and allow for the study of the cellular and molecular basis of normal and abnormal cardiac morphogenesis. Recent emerging technologies of retrospective single clonal analyses make the study of cardiac morphogenesis at single cell resolution feasible. However, tissue opacity and light scattering of the heart as imaging depth is increased hinder whole-heart imaging at single cell resolution. To overcome these obstacles, a whole-embryo clearing system that can render the heart highly transparent for both illumination and detection must be developed. Fortunately, in the last several years, many methodologies for whole-organism clearing systems such as CLARITY, Scale, SeeDB, ClearT, 3DISCO, CUBIC, DBE, BABB and PACT have been reported. This lab is interested in the cellular and molecular mechanisms of cardiac morphogenesis. Recently, we established single cell lineage tracing via the ROSA26-CreERT2; ROSA26-Confetti system to sparsely label cells during cardiac development. We adapted several whole embryo-clearing methodologies including Scale and CUBIC (clear, unobstructed brain imaging cocktails and computational analysis) to clear the embryo in combination with whole mount staining to image single clones inside the heart. The heart was successfully imaged at single cell resolution. We found that Scale can clear the embryonic heart, but cannot effectively clear the postnatal heart, while CUBIC can clear the postnatal heart, but damages the embryonic heart by dissolving the tissue. The methods described here will permit the study of gene function at a single clone resolution during cardiac morphogenesis, which, in turn, can reveal the cellular and molecular basis of congenital heart defects.

We describe a protocol to volumetrically image fluorescent protein labeled cells deep inside intact embryonic and postnatal hearts. Utilizing tissue-clearing methods in combination with whole mount staining, single fluorescent protein-labeled cells inside an embryonic or postnatal heart can be imaged clearly and accurately.

Single clonal tracing and analysis at the whole-heart level can determine cardiac progenitor cell behavior and differentiation during cardiac development, ...

Analysis of Retinoic Acid-induced Neural Differentiation of Mouse Embryonic Stem Cells in Two and Three-dimensional Embryoid Bodies

Mouse embryonic stem cells (ESCs) isolated from the inner mass of the blastocyst (typically at day E3.5), can be used as in vitro model system for studying early embryonic development. In the absence of leukemia inhibitory factor (LIF), ESCs differentiate by default into neural precursor cells. They can be amassed into a three dimensional (3D) spherical aggregate termed embryoid body (EB) due to its similarity to the early stage embryo. EBs can be seeded on fibronectin-coated coverslips, where they expand by growing two dimensional (2D) extensions, or implanted in 3D collagen matrices where they continue growing as spheroids, and differentiate into the three germ layers: endodermal, mesodermal, and ectodermal. The 3D collagen culture mimics the in vivo environment more closely than the 2D EBs. The 2D EB culture facilitates analysis by immunofluorescence and immunoblotting to track differentiation. We have developed a two-step neural differentiation protocol. In the first step, EBs are generated by the hanging-drop technique, and, simultaneously, are induced to differentiate by exposure to retinoic acid (RA). In the second step, neural differentiation proceeds in a 2D or 3D format in the absence of RA.

We describe a technique for using murine embryonic stem cells for generating two or three dimensional embryoid bodies. We then explain how to induce neural differentiation of the embryoid body cells by retinoic acid, and how to analyze their state of differentiation by progenitor cell marker immunofluorescence and immunoblotting.

Mouse embryonic stem cells (ESCs) isolated from the inner mass of the blastocyst (typically at day E3.5), can be used as in vitro model system for ...

Live Imaging of Mouse Secondary Palate Fusion

The fusion of the secondary palatal shelves to form the intact secondary palate is a key process in mammalian development and its disruption can lead to cleft secondary palate, a common congenital anomaly in humans. Secondary palate fusion has been extensively studied leading to several proposed cellular mechanisms that may mediate this process. However, these studies have been mostly performed on fixed embryonic tissues at progressive timepoints during development or in fixed explant cultures analyzed at static timepoints. Static analysis is limited for the analysis of dynamic morphogenetic processes such a palate fusion and what types of dynamic cellular behaviors mediate palatal fusion is incompletely understood. Here we describe a protocol for live imaging of ex vivo secondary palate fusion in mouse embryos. To examine cellular behaviors of palate fusion, epithelial-specific Keratin14-cre was used to label palate epithelial cells in ROSA26-mTmGflox reporter embryos. To visualize filamentous actin, Lifeact-mRFPruby reporter mice were used. Live imaging of secondary palate fusion was performed by dissecting recently-adhered secondary palatal shelves of embryonic day (E) 14.5 stage embryos and culturing in agarose-containing media on a glass bottom dish to enable imaging with an inverted confocal microscope. Using this method, we have detected a variety of novel cellular behaviors during secondary palate fusion. An appreciation of how distinct cell behaviors are coordinated in space and time greatly contributes to our understanding of this dynamic morphogenetic process. This protocol can be applied to mutant mouse lines, or cultures treated with pharmacological inhibitors to further advance understanding of how secondary palate fusion is controlled.

Here, we present a protocol for live imaging of mouse secondary palate fusion using confocal microscopy. This protocol can be used in combination with a variety of fluorescent reporter mouse lines, and with pathway inhibitors for mechanistic insight. This protocol can be adapted for live imaging in other developmental systems.

The fusion of the secondary palatal shelves to form the intact secondary palate is a key process in mammalian development and its disruption can lead to ...

A Novel Use of Three-dimensional High-frequency Ultrasonography for Early Pregnancy Characterization in the Mouse

High-frequency ultrasonography (HFUS) is a common method to non-invasively monitor the real-time development of the human fetus in utero. The mouse is routinely used as an in vivo model to study embryo implantation and pregnancy progression. Unfortunately, such murine studies require pregnancy interruption to enable follow-up phenotypic analysis. To address this issue, we used three-dimensional (3-D) reconstruction of HFUS imaging data for early detection and characterization of murine embryo implantation sites and their individual developmental progression in utero. Combining HFUS imaging with 3-D reconstruction and modeling, we were able to accurately quantify embryo implantation site number as well as monitor developmental progression in pregnant C57BL6J/129S mice from 5.5 days post coitus (d.p.c.) through to 9.5 d.p.c. with the use of a transducer. Measurements included: number, location, and volume of implantation sites as well as inter-implantation site spacing; embryo viability was assessed by cardiac activity monitoring. In the immediate post-implantation period (5.5 to 8.5 d.p.c.), 3-D reconstruction of the gravid uterus in both mesh and solid overlay format enabled visual representation of the developing pregnancies within each uterine horn. As genetically engineered mice continue to be used to characterize female reproductive phenotypes derived from uterine dysfunction, this method offers a new approach to detect, quantify, and characterize early implantation events in vivo. This novel use of 3-D HFUS imaging demonstrates the ability to successfully detect, visualize, and characterize embryo-implantation sites during early murine pregnancy in a non-invasive manner. The technology offers a significant improvement over current methods, which rely on the interruption of pregnancies for gross tissue and histopathologic characterization. Here we use a video and text format to describe how to successfully perform ultrasounds of early murine pregnancy to generate reliable and reproducible data with reconstruction of the uterine form in mesh and solid 3-D images.

Mice are widely used to study gestational biology. However, pregnancy termination is required for such studies which precludes longitudinal investigations and necessitates the use of large numbers of animals. Therefore, we describe a non-invasive technique of high-frequency ultrasonography for early detection and monitoring of post-implantation events in the pregnant mouse.

High-frequency ultrasonography (HFUS) is a common method to non-invasively monitor the real-time development of the human fetus in utero. The mouse is ...

Visualization of Cellular Electrical Activity in Zebrafish Early Embryos and Tumors

Bioelectricity, endogenous electrical signaling mediated by ion channels and pumps located on the cell membrane, plays important roles in signaling processes of excitable neuronal and muscular cells and many other biological processes, such as embryonic developmental patterning. However, there is a need for in vivo electrical activity monitoring in vertebrate embryogenesis. The advances of genetically encoded fluorescent voltage indicators (GEVIs) have made it possible to provide a solution for this challenge. Here, we describe how to create a transgenic voltage indicator zebrafish using the established voltage indicator, ASAP1 (Accelerated Sensor of Action Potentials 1), as an example. The Tol2 kit and a ubiquitous zebrafish promoter, ubi, were chosen in this study. We also explain&#160;the processes of Gateway site-specific cloning, Tol2 transposon-based zebrafish transgenesis, and the imaging process for early-stage fish embryos and fish tumors using regular epifluorescent microscopes. Using this fish line, we found that there are cellular electric voltage changes during zebrafish embryogenesis, and fish larval movement. Furthermore, it was observed that in a few zebrafish malignant peripheral nerve sheath tumors, the tumor cells were generally polarized compared to the surrounding normal tissues.

Here, we show the process of creating a cellular electric voltage reporter zebrafish line to visualize embryonic development, movement, and fish tumor cells in vivo.

Bioelectricity, endogenous electrical signaling mediated by ion channels and pumps located on the cell membrane, plays important roles in signaling ...

Whole-mount Confocal Microscopy for Adult Ear Skin: A Model System to Study Neuro-vascular Branching Morphogenesis and Immune Cell Distribution

Here, we present a protocol of a whole-mount adult ear skin imaging technique to study comprehensive three-dimensional neuro-vascular branching morphogenesis and patterning, as well as immune cell distribution at a cellular level. The analysis of peripheral nerve and blood vessel anatomical structures in adult tissues provides some insights into the understanding of functional neuro-vascular wiring and neuro-vascular degeneration in pathological conditions such as wound healing. As a highly informative model system, we have focused our studies on adult ear skin, which is readily accessible for dissection. Our simple and reproducible protocol provides an accurate depiction of the cellular components in the entire skin, such as peripheral nerves (sensory axons, sympathetic axons, and Schwann cells), blood vessels (endothelial cells and vascular smooth muscle cells), and inflammatory cells. We believe this protocol will pave the way to investigate morphological abnormalities in peripheral nerves and blood vessels as well as the inflammation in the adult ear skin under different pathological conditions.

Here, we describe a high resolution whole-mount imaging method in the entire adult mouse ear skin, which enables us to visualize branching morphogenesis and patterning of peripheral nerves and blood vessels, as well as immune cell distribution.